Rewriting the genetic code

doi:10.1126/science.abi9892

GSTDTAP > 气候变化

DOI	10.1126/science.abi9892
	Rewriting the genetic code
	Delilah Jewel; Abhishek Chatterjee
	2021-06-04
发表期刊	Science
出版年	2021
英文摘要	The near-universally conserved genetic code governs the messenger RNA (mRNA)–templated synthesis of proteins in all domains of life, using just 20 amino acid building blocks. Progress has been made toward artificially expanding the genetic code to enable cotranslational, site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in living cells. ([ 1 ][1], [ 2 ][2]). Hundreds of different ncAAs have been genetically encoded in various domains of life, enabling powerful new ways to probe and manipulate protein functions. However, this approach has been largely restricted to the incorporation of a single ncAA into a polypeptide. On page 1057 of this issue, Robertson et al. ([ 3 ][3]) report site-specific incorporation of multiple distinct ncAAs into proteins with impressive efficiency and versatility, using liberated sense codons. The ability to generate designer proteins using multiple non-natural building blocks will unlock countless applications, from the development of new classes of biotherapeutics to biomaterials with innovative properties. Incorporating ncAAs into proteins in living cells involves the use of an engineered transfer RNA (tRNA)–aminoacyltRNA synthetase (aaRS) pair, which does not cross-react with its counterparts from the host cell and delivers the desired ncAA in response to a distinct codon—most frequently a reassigned nonsense (stop) codon. The inflexibility of the canonical genetic code, wherein the meaning of the 64 triplet codons is defined, has impeded advances toward simultaneous and unrestricted incorporation of multiple different ncAAs. Only the three nonsense codons are available for reassignment, which inherently limits how many distinct ncAAs may be simultaneously used. Moreover, the natural function of stop codons—the release factor–mediated termination of translation—competes with ncAA incorporation, compromising the decoding efficiency. Even though it has been possible to site-specifically incorporate up to three different ncAAs by simultaneously reassigning all three stop codons, the low efficiency restricts the incorporation of each ncAA to a single site per polypeptide ([ 4 ][4]). Four-base “frameshift” codons have been explored as an alternative to nonsense codons for ncAA incorporation ([ 5 ][5]). However, competing recognition of the first three bases of such four-base codons, and inefficient processing at the ribosome relative to triplet codons, reduce their decoding efficiency. The recent development of a heritable unnatural base pair, which does not cross-pair with their natural counterparts and can be processed by endogenous transcription and translation machinery, has provided access to fundamentally new triplet codons that can be assigned to ncAAs ([ 6 ][6]). But, it is unclear whether the current version of this technology is suitable for simultaneous incorporation of multiple ncAAs in an unrestricted number of sites. ![Figure][7] Encoding designer proteins The genetic code of Escherichia coli was engineered to abolish the use of two sense codons (UCA and UCG) and a nonsense codon (UAG). This freed these codons up for incorporating up to three different noncanonical amino acids (ncAAs) at the same time when these codons appear in messenger RNA. GRAPHIC: V. ALTOUNIAN/ SCIENCE There is a high degree of redundancy within the canonical genetic code. For example, three codons are assigned to translation termination, whereas six codons are assigned to each of the amino acids serine, leucine, and arginine. Reconfiguring the genetic code to partially reduce this redundancy provides an attractive avenue to liberate some of the triplet codons for ncAAs. However, doing so demands global genome engineering to remove all instances of the chosen triplet codon(s), as well as the mechanism of their recognition—a truly daunting task. This was first demonstrated in Escherichia coli , in which all UAG nonsense codons were replaced with UAA, using an iterative recombination-mediated site-directed mutagenesis strategy ([ 7 ][8]). Subsequent deletion of release factor 1 eliminated endogenous recognition of UAG, which significantly enhanced the efficiency of ncAA incorporation at this codon. ([ 8 ][9], [ 9 ][10]). This work demonstrated the advantage of assigning ncAAs to natural triplet codons that are truly “blank.” However, extending this approach to liberate sense codons, which are present in far greater numbers relative to UAG, has proved challenging ([ 10 ][11]). To overcome this limitation, Fredens et al. developed an elegant approach for efficient global engineering of the E. coli genome, called REXER (replicon excision for enhanced genome engineering through programmed recombination) ([ 11 ][12]). This involves precise excision of large segments of the genome using CRISPR-Cas9, followed by their efficient substitution with synthetic counterparts using recombination. With this approach, the authors engineered E. coli strain Syn61, in which all instances of serine codons UCG and UCA, and the stop codon UAG, were substituted by their synonyms AGC, AGU, and UAA, respectively. This tour-de-force genome engineering effort, involving .18,000 codon changes, transforms UCG, UCA, and UAG to blank codons, once the corresponding cognate tRNAs, serT and serU , and release factor 1 are removed from the genome to create the strain Syn61.Δ3. Robertson et al. used the Syn61.Δ3 strain to demonstrate the advantage of assigning ncAAs to sense codons, enabling multisite ncAA incorporation with improved efficiency and versatility (see the figure). Syn61.Δ3 was evolved to create Syn61.Δ3(ev5), a mutant strain with significantly improved growth kinetics. This strain was resistant to a cocktail of different bacteriophages (viruses that infect bacteria) because of its inability to process UCG, UCA, and UAG. The authors then used archaea-derived tyrosyl and pyrrolysyl pairs—developed previously for expanding the genetic code of E. coli —to incorporate ncAAs in response to the liberated sense codons. This enabled efficient incorporation of two distinct ncAAs at up to three different sites each and the simultaneous incorporation of three distinct ncAAs in response to UCG, UCA, and UAG. It has long been hypothesized that liberating a subset of sense codons for reassignment could improve the robustness and versatility of genetic-code expansion technology. This work elegantly transforms that dream into a reality and renders validity to these hypotheses. Collectively, the development and application of the Syn61.Δ3 strain provides a blueprint for further compression of the genetic code and liberation of additional sense codons. Coupled with the development of mutually orthogonal tRNA-aaRS pairs to efficiently reassign these codons, such efforts will make it possible to incorporate many distinct ncAAs into proteins with unprecedented versatility and efficiency. This will enable countless applications, including the ribosomal synthesis of sequence-defined, genetically encoded non-natural biopolymers. The ability to generate and evolve such non-natural biopolymers with the same versatility as polypeptides could have broad implications for disciplines ranging from medicine to materials science. 1. [↵][13]1. J. W. Chin , Nature 550, 53 (2017). [OpenUrl][14][CrossRef][15][PubMed][16] 2. [↵][17]1. D. D. Young, 2. P. G. Schultz , ACS Chem. Biol. 13, 854 (2018). [OpenUrl][18][CrossRef][19][PubMed][20] 3. [↵][21]1. W. E. Robertson et al ., Science 372, 1057 (2021). [OpenUrl][22][Abstract/FREE Full Text][23] 4. [↵][24]1. J. S. Italia et al ., J. Am. Chem. Soc. 141, 6204 (2019). [OpenUrl][25][CrossRef][26][PubMed][27] 5. [↵][28]1. H. Neumann, 2. K. Wang, 3. L. Davis, 4. M. Garcia-Alai, 5. J. W. Chin , Nature 464, 441 (2010). [OpenUrl][29][CrossRef][30][PubMed][31][Web of Science][32] 6. [↵][33]1. Y. Zhang et al ., Nature 551, 644 (2017). [OpenUrl][34][CrossRef][35][PubMed][36] 7. [↵][37]1. M. J. Lajoie et al ., Science 342, 357 (2013). [OpenUrl][38][Abstract/FREE Full Text][39] 8. [↵][40]1. M. Amiram et al ., Nat. Biotechnol. 33, 1272 (2015). [OpenUrl][41][CrossRef][42][PubMed][43] 9. [↵][44]1. Y. Zheng et al ., Mol. Biosyst. 12, 1746 (2016). [OpenUrl][45] 10. [↵][46]1. N. Ostrov et al ., Science 353, 819 (2016). [OpenUrl][47][Abstract/FREE Full Text][48] 11. [↵][49]1. J. Fredens et al ., Nature 569, 514 (2019). [OpenUrl][50][CrossRef][51][PubMed][52] Acknowledgments: The authors are supported by the National Institute of General Medical Sciences (NIGMS, R35GM136437). A.C. is a senior advisor at BrickBio and owns equity therein. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5 [6]: #ref-6 [7]: pending:yes [8]: #ref-7 [9]: #ref-8 [10]: #ref-9 [11]: #ref-10 [12]: #ref-11 [13]: #xref-ref-1-1 "View reference 1 in text" [14]: {openurl}?query=rft.jtitle%253DNature%26rft.volume%253D550%26rft.spage%253D53%26rft_id%253Dinfo%253Adoi%252F10.1038%252Fnature24031%26rft_id%253Dinfo%253Apmid%252F28980641%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [15]: /lookup/external-ref?access_num=10.1038/nature24031&link_type=DOI [16]: /lookup/external-ref?access_num=28980641&link_type=MED&atom=%2Fsci%2F372%2F6546%2F1040.atom [17]: #xref-ref-2-1 "View reference 2 in text" [18]: {openurl}?query=rft.jtitle%253DACS%2BChem.%2BBiol.%26rft.volume%253D13%26rft.spage%253D854%26rft_id%253Dinfo%253Adoi%252F10.1021%252Facschembio.7b00974%26rft_id%253Dinfo%253Apmid%252F29345901%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [19]: /lookup/external-ref?access_num=10.1021/acschembio.7b00974&link_type=DOI [20]: /lookup/external-ref?access_num=29345901&link_type=MED&atom=%2Fsci%2F372%2F6546%2F1040.atom [21]: #xref-ref-3-1 "View reference 3 in text" [22]: {openurl}?query=rft.jtitle%253DScience%26rft.stitle%253DScience%26rft.aulast%253DRobertson%26rft.auinit1%253DW.%2BE.%26rft.volume%253D372%26rft.issue%253D6546%26rft.spage%253D1057%26rft.epage%253D1062%26rft.atitle%253DSense%2Bcodon%2Breassignment%2Benables%2Bviral%2Bresistance%2Band%2Bencoded%2Bpolymer%2Bsynthesis%26rft_id%253Dinfo%253Adoi%252F10.1126%252Fscience.abg3029%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [23]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6Mzoic2NpIjtzOjU6InJlc2lkIjtzOjEzOiIzNzIvNjU0Ni8xMDU3IjtzOjQ6ImF0b20iO3M6MjM6Ii9zY2kvMzcyLzY1NDYvMTA0MC5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30= [24]: #xref-ref-4-1 "View reference 4 in text" [25]: {openurl}?query=rft.jtitle%253DJ.%2BAm.%2BChem.%2BSoc.%26rft.volume%253D141%26rft.spage%253D6204%26rft_id%253Dinfo%253Adoi%252F10.1021%252Fjacs.8b12954%26rft_id%253Dinfo%253Apmid%252F30909694%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [26]: /lookup/external-ref?access_num=10.1021/jacs.8b12954&link_type=DOI [27]: /lookup/external-ref?access_num=30909694&link_type=MED&atom=%2Fsci%2F372%2F6546%2F1040.atom [28]: #xref-ref-5-1 "View reference 5 in text" [29]: {openurl}?query=rft.jtitle%253DNature%26rft.stitle%253DNature%26rft.aulast%253DNeumann%26rft.auinit1%253DH.%26rft.volume%253D464%26rft.issue%253D7287%26rft.spage%253D441%26rft.epage%253D444%26rft.atitle%253DEncoding%2Bmultiple%2Bunnatural%2Bamino%2Bacids%2Bvia%2Bevolution%2Bof%2Ba%2Bquadruplet-decoding%2Bribosome.%26rft_id%253Dinfo%253Adoi%252F10.1038%252Fnature08817%26rft_id%253Dinfo%253Apmid%252F20154731%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [30]: /lookup/external-ref?access_num=10.1038/nature08817&link_type=DOI [31]: /lookup/external-ref?access_num=20154731&link_type=MED&atom=%2Fsci%2F372%2F6546%2F1040.atom [32]: /lookup/external-ref?access_num=000275657100053&link_type=ISI [33]: #xref-ref-6-1 "View reference 6 in text" [34]: {openurl}?query=rft.jtitle%253DNature%26rft.volume%253D551%26rft.spage%253D644%26rft_id%253Dinfo%253Adoi%252F10.1038%252Fnature24659%26rft_id%253Dinfo%253Apmid%252F29189780%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [35]: /lookup/external-ref?access_num=10.1038/nature24659&link_type=DOI [36]: /lookup/external-ref?access_num=29189780&link_type=MED&atom=%2Fsci%2F372%2F6546%2F1040.atom [37]: #xref-ref-7-1 "View reference 7 in text" [38]: 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{openurl}?query=rft.jtitle%253DNat.%2BBiotechnol.%26rft.volume%253D33%26rft.spage%253D1272%26rft_id%253Dinfo%253Adoi%252F10.1038%252Fnbt.3372%26rft_id%253Dinfo%253Apmid%252F26571098%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [42]: /lookup/external-ref?access_num=10.1038/nbt.3372&link_type=DOI [43]: /lookup/external-ref?access_num=26571098&link_type=MED&atom=%2Fsci%2F372%2F6546%2F1040.atom [44]: #xref-ref-9-1 "View reference 9 in text" [45]: {openurl}?query=rft.jtitle%253DMol.%2BBiosyst.%26rft.volume%253D12%26rft.spage%253D1746%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [46]: #xref-ref-10-1 "View reference 10 in text" [47]: {openurl}?query=rft.jtitle%253DScience%26rft.stitle%253DScience%26rft.aulast%253DOstrov%26rft.auinit1%253DN.%26rft.volume%253D353%26rft.issue%253D6301%26rft.spage%253D819%26rft.epage%253D822%26rft.atitle%253DDesign%252C%2Bsynthesis%252C%2Band%2Btesting%2Btoward%2Ba%2B57-codon%2Bgenome%26rft_id%253Dinfo%253Adoi%252F10.1126%252Fscience.aaf3639%26rft_id%253Dinfo%253Apmid%252F27540174%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [48]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6Mzoic2NpIjtzOjU6InJlc2lkIjtzOjEyOiIzNTMvNjMwMS84MTkiO3M6NDoiYXRvbSI7czoyMzoiL3NjaS8zNzIvNjU0Ni8xMDQwLmF0b20iO31zOjg6ImZyYWdtZW50IjtzOjA6IiI7fQ== [49]: #xref-ref-11-1 "View reference 11 in text" [50]: {openurl}?query=rft.jtitle%253DNature%26rft.volume%253D569%26rft.spage%253D514%26rft_id%253Dinfo%253Adoi%252F10.1038%252Fs41586-019-1192-5%26rft_id%253Dinfo%253Apmid%252F31092918%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [51]: /lookup/external-ref?access_num=10.1038/s41586-019-1192-5&link_type=DOI [52]: /lookup/external-ref?access_num=31092918&link_type=MED&atom=%2Fsci%2F372%2F6546%2F1040.atom
领域	气候变化 ; 资源环境
URL	查看原文
引用统计
文献类型	期刊论文
条目标识符	http://119.78.100.173/C666/handle/2XK7JSWQ/329863
专题	气候变化资源环境科学
推荐引用方式 GB/T 7714	Delilah Jewel,Abhishek Chatterjee. Rewriting the genetic code[J]. Science,2021.
APA	Delilah Jewel,&Abhishek Chatterjee.(2021).Rewriting the genetic code.Science.
MLA	Delilah Jewel,et al."Rewriting the genetic code".Science (2021).